The invention relates to a method for identification of Escherichia coli (E. coli) strain DSM 6601.
Escherichia coli is a gram-negative bacterium that occurs in human and animal intestinal flora as well as outside the intestines. Among the microbial cloning systems of genetic engineering, E. coli is now the most important host organism for expression of heterologous proteins as well as for cloning and DNA amplification.
E. coli exists in numerous varieties, which differ as regards capsule antigens (K antigens), surface antigens (O antigens) and flagella antigens (H antigens) and can therefore be subdivided into numerous serological types. Classification by serotypes, however, does not provide any indication of the different virulence of the pathogens. Representatives of one and the same serotype can have different pathogenic potential both in the human and in the animal body, ranging in the extreme case from avirulent to highly pathogenic. It is known that E. coli strain DSM 6601 is rated as nonpathogenic to humans or animals.
Thus there still exists a need for methods of verification of nonpathogenic E. coli strains. Serotyping is not adequate as the only method for evaluating whether an E. coli strain is pathogenic or nonpathogenic. It has already been mentioned that both pathogenic and nonpathogenic variants occur under the same serotype. For diagnostic and therapeutic purposes in medicine, and also for use for genetic engineering purposes, the ability unequivocally to identify individual strains is therefore desirable.
According to the invention, a method for identification of E. coli strain DSM 6601 is now proposed which is characterized in that certain primer pairs from the plasmids or from the fimA and focA sequences of the bacterial DNA are used in a PCR reaction.
PCR (polymerase chain reaction) is a method in which a few molecules of an arbitrary genomic DNA sequence can be multiplied in vitro by factors of 106 to 108 in extremely short time. The detection method according to the invention is based on the method described by R. K. Saiki et al. in Science 239: 487491 (1988).
PCR is performed by using primers, or in otherwise oligonucleotides, which usually have a length of about 15 to 30 nucleotides and the sequences of which are complementary to the initial or terminal sequences of the sister strands of the DNA to be amplified.
The double-stranded DNA of the sequence to be amplified is first denatured by heating, thus splitting it into individual strands. At a later stage, the complementary strand will be formed over the single-stranded region of the nucleic acid known as the template or matrix. After denaturing by heating, the mixture containing the primers is cooled, during which the primer nucleotides at the ends of the single-stranded DNA are hybridized and thus prevent recombination of the original single DNA strands. The temperature is then raised and a mixture of the four nucleotide-5xe2x80x2-triphosphates typical of DNA is added, as is a thermally stable DNA polymerase. Taq polymerase from the extremely thermophilic organism Thermus aquaticus, which even survives brief heating to above 95xc2x0 C., has been found to be particularly suitable. At 72xc2x0 C., the single DNA strand between the two ends occupied by primers is made up to a double strand by the polymerase.
The three process steps, namely denaturing by heat, primer annealing and polymerization, can be repeated until the mixture is exhausted. Since doubling of the DNA quantity is achieved in each individual step, a multiplication factor of about 106 is theoretically achieved after about 20 cycles.
In the present invention, there are used as primer pairs such from the fimA sequence (SEQ ID NO:1) designated Muta 1 (SEQ ID NO:7) and 2 (SEQ ID NO:8) (FIG. 1) and such from the focA sequence (SEQ ID NO:2) designated Muta 3 (SEQ ID NO:9) and 4 (SEQ ID NO:10) (FIG. 2) of strain DSM 6601. These DNA sequences are identical in parts to genes of other enterobacteria, although at some positions they contain bases which have not yet been observed there in other enterobacteria.
The further primer pairs Muta 5 (SEQ ID NO:11) and 6 (SEQ ID NO:12) (FIG. 3), Muta 7 (SEQ ID NO:13) and 8 (SEQ ID NO:14) as well as Muta 9 (SEQ ID NO:15) and 10 (SEQ ID NO:16) (FIG. 4) were selected from the DNA sequences of the plasmids PMUT 1 (SEQ ID NO:3) (FIG. 5) and pMUT 2 (SEQ ID NO:4) (FIG. 6) of strain DSM 6601. These primer pairs also exhibit a nucleotide sequence which heretofore has not been found as such in enterobacteria.
The sequences of the primers Muta 1 to Muta 10 (SEQ ID NOS:7-16) are illustrated in detail in the attached FIGS. 1 to 4.